Oligonucleotides that can be used in the amplification and detection of CMV nucleic acid

ABSTRACT

The sensitivity and reliability (robustness) of CMV mRNA detection is greatly dependent on the selection of suitable oligonucleotides for amplification, since there is sequence variation among strains of CMV potentially in every region of the genome. The present invention is concerned with oligonucleotides that can be used in the amplification and detection of human Cytomegalovirus (HCMV) mRNA. These novel oligonucleotides show an improved sensitivity and robustness of CMV mRNA detection if compared with known sequences when used in amplification and detection. Furthermore a method for the diagnosis of HCMV disease is provided.

This application is a continuation-in-part of Ser. No. 08/628,654, filedMar. 27, 1996, abandoned.

FIELD OF THE INVENTION

The present invention is concerned with oligonucleotides that can beused in the amplification detection of human Cytomegalovirus (HCMV)mRNA. Furthermore a method for the diagnosis of HCMV disease isprovided.

BACKGROUND OF THE INVENTION

Human Cytomegalovirus is an ubiquitous Herpes-type virus, having adouble stranded DNA genome of about 240,000 nucleotides in length thatinfects 40-80% of humans before puberty. A prominent feature common toall herpesviruses is their establishment of lifelong persistence afterinfection and their ability to cause recurrent infection afterreactivation (Stevens, J. G.. Microbiol. Rev. 53, 318-332., 1989). HCMValso becomes latent after primary infection which often occurs withoutclinical symptoms. Even recurrent infection in most cases goesasymptomatic or leads to only mild disease in the immunocompetent host.However, in congenitally infected infants and immunocompromisedpatients, such as allograft recipients (Meyers, J. D., et al., J.Infect. Dis. 153, 478-488., 1986) or AIDS patients (Drew, W. L. JInfect. Dis 158, 449-456., 1988; Drew, W. L. Clin. Infect. Dis 14,608-615., 1992), where the fine balance between the immune system andthe latently existing virus is disturbed, HCMV may cause severe andsometimes life-threatening disease, including retinitis,gastrointestinal disorders, and encephalitis (Drew, 1992). Earlyadministration of antiviral drugs like ganciclovir and foscarnet canhave significant beneficial effects on the prognosis of a patient (Jahn,G. et al., Intervirology 35, 60-72., 1993; Schmidt, G. M. et al., N.Engl. J Med. 324, 1005-1011., 1991). Therefore, with the availability ofclinically effective antiviral therapy, early and sensitive diagnosis isof significant importance.

CMV specific antibodies, in particular IgM antibodies, can be used as amarker for CMV infection, but are of limited value when it comes todiscrimination between latent and active infections. Most viraldetection methods currently employed do not unambiguously allow forprediction of whether a given infection will be symptomatic. Furthermoreserological methods are indirect and often lack sensitivity. Viralculture is a more direct diagnostic parameter for CMV viremia. AlthoughCMV culture from blood cells appeared to be indicative for an active CMVinfection, the method does not enable rapid diagnosis and is technicallydifficult. Moreover, viral culture does not necessarily correspond toHCMV disease. A reliable relation between virus isolation fromperipheral leukocytes and the appearance of clinical symptoms may notexist in some immunosuppressed patients (Delgado, R. et al., J Clin.Microbiol. 30, 1876-1878., 1992). Also urinary or pharyngeal shedding ofthe virus frequently occurs without clinical symptoms and organinvolvement. Amplification of HCMV DNA in peripheral leukocytes bypolymerase chain reaction (PCR), although a very sensitive technique forCMV viremia, is not usable as a marker of clinically symptomatic HCMVinfection either. Due to the high sensitivity of enzymaticamplification, occasionally HCMV DNA was detectable in peripheralleukocytes without HCMV-related disease. Latent viral genomes may bedetected by this technique or a patient may remain HCMV-DNA positiveover a prolonged period of time after the disease has resolved (Jahn, G.et al., 1993, Zipeto, D. et al., J Clin. Microbiol. 30, 527-530., 1992;Delgado et al., 1992).

At the moment, the method of choice for the early diagnosis of acutesymptomatic HCMV infection is the antigenemia assay based onimmunological detection of the structural protein pp65 by using specificantibodies (Storch, G. A., et al., J. Clin. Microbiol. 32, 997-1003.,1994; Gerna, G. et al., J. Infect. Dis. 164, 488-498., 1991; Gerna, G.,et al., J Clin. Microbiol. 30, 1232-1237.98., 1992). However, a matterof concern employing this method is its sensitivity. The number ofpp65-positive cells in the early course of infection may be very low.Furthermore, in expressing cells stability of the pp65 antigen appearedto be limited (Chou, S., Curr. Opin. Infect. Dis. 5, 427-432., 1991) andsensitivity can be reduced due to the application of monoclonalantibodies rather than a pool of anti-pp65 antibodies that wouldrecognize different epitopes of the protein.

Since viral replication requires transcription of mRNA species, the useof HCMV mRNA detection as a marker for active CMV infection wasinvestigated (Bitsch, A. et al., J Infect. Dis 167, 740-743, 1993.

Recently, HCMV infections were examined on the transcript level usingRNA amplification (Bitsch, A. et al., 1993; Meyer, T. et al., Mol. CellProbes. 8, 261-271., 1994; Gerna, G., et al., J Clin. Microbiol. 30,1232-1237.98, 1993; Gerna, G., et al., J Clin. Microbiol. 30,1232-1237.98, 1992). In principle, like detection of viral antigens,analysis of viral transcripts expressed in association with viralreplication should allow reliable diagnosis of symptomatic infections.

More recently, the detection of certain mRNA's of HCMV i.e. based on IEA(immediate early antigen) and the matrix tegument protein pp67 mRNA havebeen described in US Serial No. 08/628,654. However, the noveloligonucleotides of the present invention have several advantages overthe oligonucleotides as disclosed in the previous application.Comparison studies are presented in the experimental part of thedescription.

The sensitivity and reliability (robustness) of CMV mRNA detection isgreatly dependent on the selection of the oligonucleotides used in theamplification, since there is sequence variation among strains of CMVpotentially in every region of the genome. Ideally, primer selectionshould be based on knowledge of interstrain variability in candidateprimer sequences and the consequences of mismatching at primer sites.(Chou S., J. of Clin. Microbiol., 2307-2310, 1992).

Therefore, the need exists for suitable oligonucleotides includingnucleic acid sequences that can be used as in the amplification andsubsequent detection of all strain variants of CMV.

SUMMARY OF THE INVENTION

The present invention is related to the detection of a certain late HCMVmRNA and provides oligonucleotides suitable for use in the amplificationand subsequent detection of this mRNA. The binding sites of theoligonucleotides according to the present invention are located in thematrix tegument protein pp67 encoding gene sequence (UL65), which isexpressed during the late phase of CMV infection.

Preferred oligonucleotides according to the present invention,correspond to part of a nucleic acid sequence encoding HCMV pp67, saidoligonucleotide being 10-35 nucleotides in length and comprising atleast a fragment of 10 nucleotides of a sequence selected from the groupconsisting of:

5'-GGGTCGATTCAGACTGA-3' [SEQ.ID.No.: 10];

5'-CTGGAGATATATGTTGACCA-3' [SEQ.ID.No.: 11],

5'-GGATTCGGACTTTCCGTTCGA-3' [SEQ.ID.No.: 8], and

5'-CCAAAAAGCTAGCCGTCACG-3' [SEQ.ID.No.: 12], or its complementarysequence.

A preferred embodiment of the present invention is directed to anoligonucleotide linked to a promoter sequence.

DETAILED DESCRIPTION OF THE INVENTION

An example of oligonucleotide pairs according to the present inventionincludes the following oligonucleotide sequences:5'-aattctaatacgactcactatagggagaGGGTCGATTCAGACTGA-3' [SEQ ID NO: 9] or5'-aattctaatacgactcactatagggagaGGGTCGATTCGAGACCGA-3' [SEQ ID NO: 5] incombination with 5'-GACCTGATATCCCTCCATATA-3' [SEQ ID NO: 7]. (The T7promoter sequence is shown, but may be replaced by any other suitablepromoter sequence.) A probe that may be used for the detection of theamplificate generated using this pair may comprise an oligonucleotideconsisting essentially of the following sequence:5'-GGATTCGGACTTTCCGTTCGA-3' [SEQ ID NO: 8]. Probes comprising saidsequence are also part of the present invention.

For the pp67 gene sequence interstrain variations between CMV AD169 andTowne exist. Therefore, for this target two oligonucleotide pairs werechosen, CMV-pp67-1 and CMV-pp67-4 (Table 1), either of which was derivedfrom the same region of the gene but each based on a differentlaboratory strain. An example of oligonucleotides for the detection ofpp67 mRNA are oligonucleotides, 10-35 nucleotides in length comprising,at least a fragment of 10 nucleotides, of a sequence selected from thegroup consisting of:

5'-GGGTCGATTCAGACTGA-3' [SEQ ID NO:10]

5'-GGGTCGATTCGAGACCGA-3' [SEQ ID NO: 6]; and

5'-GACCTGATATCCCTCCATATA-3' [SEQ ID NO: 7].

A preferred embodiment of the present invention is directed to thefollowing oligonucleotide sequences:5'-aattctaatacgactcactatagggagaGGGTCGATTCAGACTGA-3' [SEQ ID NO: 9] incombination with 5'-CTGGAGATATATGTTGACCA-3' [SEQ ID NO:11], for theamplification. (The T7 promoter sequence is shown, but may be replacedby any other suitable promoter sequence.) In this preferred embodimentof the present invention, an oligonucleotide consisting essentially ofthe sequence 5'-GGATTCGGACTTTCCGTTCGA-3' [SEQ ID NO: 8] or5'-CCAAAAAGCTAGCCGTCACG-3' [SEQ ID NO:12], provided with a detectablelabel, is the the preferred probe for the detection of the amplificategenerated using the preferred oligonucleotides according to the presentinvention.

An oligonucleotide sequence used as a detection probe may be labeledwith a detectable moiety. Various labeling moieties are known in theart. Said moiety may, for example, either be a radioactive compound, adetectable enzyme (e.g. horse radish peroxidase (HRP)) or any othermoiety capable of generating a detectable signal such as a calorimetric,fluorescent, chemiluminescent or electrochemiluminescent signal.Preferred analysis systems wherein said labels are used areelectrochemiluminescence (ECL) based analysis or enzyme linked gel assay(ELGA) based analysis.

As already indicated above, and will be presented in the experimentalpart of the description, both the sensitivity and reliability of CMVmRNA detection is greatly improved using the oligonucleotides accordingto the present invention when compared to known oligonucleotides used inthis art.

The term "oligonucleotide" as used herein refers to a molecule comprisedof two or more deoxyribonucleotides or ribonucleotides such as primersand probes.

The term "primer" as used herein refers to an oligonucleotide eithernaturally occurring (e.g. as a restriction fragment) or producedsynthetically, which is capable of acting as a point of initiation ofsynthesis of a primer extension product which is complementary to anucleic acid strand (template or target sequence) when placed undersuitable conditions (e.g. buffer, salt, temperature and pH) in thepresence of nucleotides and an agent for nucleic acid polymerization,such as DNA dependent or RNA dependent polymerase. A primer must besufficiently long to prime the synthesis of extension products in thepresence of an agent for polymerization. A typical primer contains atleast about 10 nucleotides in length of a sequence substantiallycomplementary (P1) or homologous (P2) to the target sequence, butsomewhat longer primers are preferred. Usually primers contain about15-26 nucleotides but longer primers may also be employed.

Normally a set of primers will consist of at least two primers, one`upstream` and one `downstream` primer which together define theamplificate (the sequence that will be amplified using said primers).

The oligonucleotides according to the invention may also be linked to apromoter sequence. The term "promoter sequence" defines a region of anucleic acid sequence that is specifically recognized by an RNApolymerase that binds to a recognized sequence and initiates the processof transcription by which an RNA transcript is produced. In principleany promoter sequence may be employed for which there is a known andavailable polymerase that is capable of recognizing the initiationsequence. Known and useful promoters are those that are recognized bycertain bacteriophage RNA polymerases such as bacteriophage T3, T7 orSP6.

It is understood that oligonucleotides consisting of the sequences ofthe present invention may contain minor deletions, additions and/orsubstitutions of nucleic acid bases, to the extent that such alterationsdo not negatively affect the yield or product obtained to a significantdegree.

Another preferred embodiment of the present invention is directed to amethod for the diagnosis of symptomatic CMV disease, wherein thepresence of pp67 mRNA encoding a late structural protein of the humanCytomegalovirus in a blood sample of an individual, suspected ofcarrying said disease, is detected, said method comprising the followingsteps:

amplifying a target sequence within said mRNA using oligonucleotidesaccording to the invention and suitable amplification reagents,

reacting the sample, optionally containing amplified nucleic acid, withan oligonucleotide according to the present invention as a detectionprobe,

detecting hybrids formed between the amplified sequence and the probe.

Various techniques for amplifying nucleic acid are known in the art. Oneexample of a technique for the amplification of a DNA target segment isthe so-called "polymerase chain reaction" (PCR). with the PCR techniquethe copy number of a particular target segment is increasedexponentially with a number of cycles. A pair of primers is used and ineach cycle a DNA primer is annealed to the 3' side of each of the twostrands of the double stranded DNA-target sequence. The primers areextended with a DNA polymerase in the presence of the variousmononucleotides to generate double stranded DNA again. The strands ofthe double stranded DNA are separated from each other by thermaldenaturation and each strand can serves as a template for primerannealing and subsequent elongation in a following cycle. The PCR methodhas been described in Saiki et al., Science 230, 135, 1985 and inEuropean Patents no. EP 200362 and EP 201184.

Another technique for the amplification of nucleic acid is thetranscription based amplification system (TAS). TAS employs anRNA-transcript-production step from a DNA, synthesized to incorporate asegment of the target sequence and a promoter, to enable transcriptionfrom the segment of a RNA with the sequence complementary to that of thetarget. Multiple cycles can be carried out as the RNA made in thetranscription step can serve as template for making similarlytranscribable DNA, which in turn, can be transcribed to yield additionalRNA. The TAS method is described in International Patent Appl. no. WO88/10315. Transcription based amplification techniques usually comprisetreating target nucleic acid with two oligonucleotides one of whichcomprises a promoter sequence, to generate a template including afunctional promoter. Multiple copies of RNA are transcribed form saidtemplate and can serve as a basis for further amplification.

An isothermal continuous transcription based amplification method is theNASBA (nucleic acid sequence based amplification) process, as describedin European Patent no. EP 329822. NASBA includes the use of T7 RNApolymerase to transcribe multiple copies of RNA from a templateincluding a T7 promoter.

For RNA amplification (as with the method according to the invention),the NASBA technology or other transcription based amplificationtechniques are preferred.

If RT-PCR is used for the detection of viral transcripts differentiationof mRNA- and DNA-derived PCR products is necessary. For splicedtranscripts, like the IEA mRNA, the exon-intron structure can be used.However, mRNA species encoding the late structural proteins are almostexclusively encoded by unspliced transcripts. DNAse treatment prior toRT-PCR can be employed (Bitsch, A. et al., J Infect. Dis 167, 740-743.,1993; Meyer, T. et al., Mol. Cell Probes. 8, 261-271., 1994), butsometimes fails to remove contaminating DNA sufficiently (Bitsch, A. etal., 1993). In contrast to RT-PCR, NASBA, which is based on RNAtranscription by T7 RNA polymerase (Kievits et al., 1991; Compton,1991), does not need differentiation between RNA- and DNA-derivedamplification products since it only uses RNA as its principal target.NASBA enables specific amplification of RNA targets even in a backgroundof DNA. Especially for unspliced targets like almost all late HCMV genetranscripts, this method is beneficial as it circumvents DNAse treatmentwhich occasionally might be inefficient (Bitsch, A. et al., 1993). Thismethod was used for the analysis of CMV transcripts in whole bloodsamples from HIV-infected individuals.

Test kits for the detection of CMV in clinical samples are also part ofthe present invention. A test kit according to the invention maycomprise a pair of oligonucleotides according to the invention and aprobe comprising an oligonucleotide according to the invention. Such atest kit may additionally comprise suitable amplification reagents suchas DNA and or RNA polymerases and mononucleotides. Test kits that can beused with the method according to the invention may comprise theoligonucleotides according to the invention for the amplification andsubsequent detection of pp67 mRNA. A preferred embodiment for the testkit comprises the oligonucleotides:5'-aattctaatacgactcactatagggagaGGGTCGATTCAGACTGA-3' [SEQ ID NO: 9] incombination with 5'-CTGGAGATATATGTTGACCA-3' [SEQ ID NO:11] for theamplification, and 5'-GGATTCGGACTTTCCGTTCGA-3' [SEQ ID NO: 8] or5'-CCAAAAAGCTAGCCGTCACG-3' [SEQ ID NO:12], provided with a detectablelabel, as probe.

The invention is further exemplified by the following examples.

EXAMPLES Example 1: Analysis of CMV DNA and mRNA in Clinical Samples

1.1 Materials and Methods

1.1.1 Clinical Specimens

Samples from patients clinically at risk of infection with CMV wereanalyzed for the presence of mRNAs encoding the immediate early antigen(IEA) or the matrix tegument protein pp67 expressed during the latephase of CMV infection.

The thirty-five blood samples were obtained from immunocompromisedpatients including 22 heart, liver, or kidney transplant recipients, 8AIDS patients, two patients with leukemia, one patient with amyelodysplastic syndrome, one patient with primary Epstein Barr virusmononucleosis, and one patient with Kaposi sarcoma. Ethylenediaminetetraacetic acid (EDTA) anticoagulated blood samples submittedconsecutively as received by the laboratory, were mixed with ninevolumes of lysis buffer [50 mM Tris-Hydrochloric acid (pH 6.4); 20 mMEDTA; 1.3% (w/v) Triton X-100; 5.25 M guanidinium thiocyanate] andstored at -70° C. until use.

1.1.2 Nucleic Acid Isolation

From the anticoagulant-treated blood specimens total nucleic acid wasisolated using guanidinium thiocyanate-mediated cell lysis andadsorption of nucleic acid to silica particles (Boom et al., J. of Clin.Microbiol. 28, 495-503, 1990).

Whole blood samples in lysis buffer were thawed and from each sample 1ml (equivalent to 100 μl whole blood) was transferred into an Eppendorftube. Subsequently, 70 μl of hydrochloric acid-activated silicum dioxideparticles [size-selected suspension of 1 mg/ml in 0.1 M Hydrochloricacid (Sigma); see ref. Boom et al., 1990] were added and the suspensionwas incubated during 10 minutes at room temperature with regularvortexing. Nucleic acid bound to the silica was spun down bycentrifugation. Pelleted silica particles were washed twice with 1 mlGuSCN wash buffer [50 mM Tris-hydrochloric acid (pH 6.4); 5.25 Mguanidinium thiocyanate], followed by two washing steps with 1 ml 70%ethanol and a single washing step with 1 ml acetone. After each washingstep, the suspension was briefly centrifuged and the silica pellet wasresuspended in the next washing solution by thorough mixing. Afterremoval of the acetone, the silica particles were dried by incubation at56° C. in a heating block during 10 minutes. Nucleic acid was elutedfrom the silica particles by incubation in 100 μl distilled water at 56°C. during 10 minutes. Finally, the silica particles were spun down againand the supernatant was carefully pipetted into fresh reaction tubesavoiding any carry-over of silica. Extracted nucleic acid samples werestored at -70° C. until use.

Prior to the detection of CMV mRNAs in these isolates, the integrity andamount of extracted RNA was validated. Therefore, samples were analyzedfor the presence of U1 snRNP-specific A protein (U1A) mRNA (Sillekens,P. T. G., et al., EMBO J. 6, 3841-3848., 1987), being a relatively lowabundant message, transcribed from a cellular housekeeping gene. Asrevealed by Northern blot analysis, presence of amplifiable U1A mRNA wasobvious in all samples (data not shown).

1.1.3 Oligonucleotides Used in Amplification and as Probes

Sequences and polarity of the oligonucleotides used in the amplificationand the probes used for specific detection, are shown in Table 1.

All oligonucleotides were synthesized on a PCR-MATE 391 DNA synthesizer(Applied Biosystems) using phosphoramidite biochemistry.Oligonucleotides for ELGA detection (see below) were synthesized with a5'-amino link (Aminolink 2; Applied Biosystems) for subsequent couplingof horseradish peroxidase (HRP).

Oligonucleotides used for amplification were purified byelectrophoretically separating the crude oligonucleotide solutions overa 20% polyacrylamide/7M urea slab gel and subsequent elution of thefull-length oligonucleotide from the corresponding gel band. Afterelution from the gel slices and concentration by ethanol precipitation,the oligonucleotides were dissolved in Milli-Q water and concentrationsdetermined by OD(260 nm) measurement.

Oligonucleotides used as detection probes were conjugated with HRP(Boehringer) by coupling the enzyme to the amino link of theoligonucleotide using the cross-linking reagents SDPD (Pharmacia) andEMCS (Fluka). Unbound HRP was removed over a Qiagen Tip-100 column(Qiagen). The HRP-labeled oligonucleotides were purified bypolyacrylamide gel electrophoresis and subsequent elution of theHRP-oligonucleotides from the gel slices by overnight incubation inwater. The amount of HRP-conjugated oligonucleotide was calculated fromOD(260 nm) and OD(400 nm) measurement. The solutions were stored at -70°C.

5'-CTGGAGATATATGTTGACCA-3'1.1.4 NASBA Amplification

RNA amplifications were performed using NASBA, since this amplificationtechnology is capable of specifically amplifying RNA in a background ofDNA. These amplification reactions were carried out using a standardNASBA protocol:

To set up an amplification reaction, 101 of 2.5×reaction buffer [100 mMTris-hydrochloric acid (pH 8.5); 30 mM magnesium chloride; 105 mMPotassium chloride; 12.5 mM dithiothreitol; 2.5 mM of each of dNTP; 5 mMof ATP, CTP and UTP; 3.75 mM of GTP; 1.25 μM of ITP] was added to areaction tube together with 6.25 μl 4×mix [0.8 μM of eacholigonucleotide used in the amplification; 60% dimethylsulphoxide], 5μlnucleic acid solution, and 1.75 μl distilled water. This mixture washeated at 65° C. during 5 minutes, after which the tubes were placed at41° C. Two μl of enzyme mix [40 units T7 RNA polymerase; 8 units AMVreverse transcriptase; 0.1 unit RNase H; 1.25 μg/μl BSA] were added andthe contents of the tube were mixed by gentle tapping. The reaction wasincubated at 41° C. for 90 minutes and stopped by placing it at -20° C.

1.1.5 Polymerase Chain Reaction

For the detection of the corresponding genes of the HCMV mRNAs PCRamplification was used (performed essentially as described in Saiki etal, 1985).

As template DNA, 5 μl nucleic acid solution were added to a total of 20μl of reaction mixture for amplification containing the appropriateoligonucleotide pair (15 pmol each), deoxyribonucleoside triphosphates(200 μM each; Pharmacia), 2 μl of 10×PCR buffer (Perkin-Elmer), and 1.25units Taq polymerase. Reactions were overlaid with 100 μl of mineral oilto prevent evaporation. The amplification was performed in a DNA thermalcycler (Perkin-Elmer) by 40 cycles of denaturation at 94° C. for 1 min,primer annealing at 60° C. for 1 min, chain extension at 72° C. for 2min, and a final extension segment at 72° C. for 10 min.

1.1.6 Southern Blot Analysis of PCR-amplified Products

Amplified DNA was transferred from a 2.0% Pronarose gel (Hispanagar,S.A.) to a nylon membrane (Zeta-probe; BioRad, USA) by vacuum blottingin 2×SSC [1×SSC is 150 mM sodium chloride; 15 mM sodium citrate] during2 hours. Membranes were preincubated at 50° C. in a hybridizationsolution [0.5 M sodium phosphate (pH 7.2); 7% sodium dodecyl sulphate]during 30 minutes prior to the addition of 32P labeled oligonucleotideprobe to a final concentration of about 105 cpm/ml. Hybridization wasperformed overnight at 50° C. Subsequent washings were carried out at50° C. in 0.3×SSC supplemented with 0.1% SDS. Autoradiography wasperformed for several hours at -70° C. with Kodak Royal X-omat film andintensifying screens.

1.1.7 Analysis of NASBA-amplified Products (ELGA)

For the analysis of NASBA products a non-radioactive enzyme linked gelassay (ELGA) based on liquid hybridization was used. Hybridization ofamplification product to a specific HRP labeled oligonucleotide probewas performed by mixing 2μl of an amplification reaction with 1 μl5×SSC, 1 μl concentrated loading buffer [25% (v/v) glycerol; 10 mMsodium phosphate buffer (pH 7.0) 0.05% bromophenol blue; 0.01% xylenecyanol], and 1 μl HRP-labeled oligonucleotide solution containing about1010 molecules per μl, followed by incubation at 45° C. during 15minutes. After hybridization, half of the reaction mixture was directlyapplied onto a 7% polyacrylamide gel supplemented with 0.04% (w/v)dextran sulphate. After separation of bound and unbound HRP-labeledoligonucleotide by electrophoresis, the probe was visualized in the gelby direct staining with 50 ml substrate solution [125 μg3,3',5,5'-tetramethylbenzidine per ml; 0.003% hydrogen peroxide; 100 mMsodium citrate buffer (pH 5.2)] for about 10 minutes at roomtemperature. Finally, the gel was fixed by overnight incubation in a 50%(v/v) methanol solution and air dried.

2.1 Results

2.1.1 Oligonucleotide Pairs and Sensitivity

To determine the analytical sensitivity attainable in NASBA with theoligonucleotide pairs a standard dilution series of in vitro generatedCMV RNA was evaluated. An RNA template of known concentration wasgenerated in vitro from a cloned subfragment of the CMV AD 169 EcoRIfragment J (Schrier, R. D., et al., Science 230, 1048-1051., 1985) forIEA and a CMV clone encompassing the pp67 gene for pp67. Standarddilution series were prepared from the in vitro generated RNA.

Sensitivity of the CMV-IEA (E4) oligonucleotide set was reproduciblyfound to be at least 100 molecules of in vitro generated RNA input inNASBA reaction. For the CMV-IEA(E2/E3) oligonucleotide pair a comparablesensitivity of 100 molecules could be achieved.

Oligonucleotide pair performance of the pp67 pairs was evaluated on invitro generated RNA transcribed from a cloned fragment of the pp67 geneof CMV AD169. For the CMV-pp67-4 oligonucleotide pair, derived fromAD169, NASBA conditions could be optimized such that the sensitivity ofthis pair was 100 molecules of in vitro generated RNA. As could beanticipated from the mismatches of the downstream primer of CMV-pp67-1,based on the Towne strain sequence, no amplification product could begenerated with this oligonucleotide pair from the CMV AD169-derived pp67RNA.

To establish whether in addition to in vitro generated RNA the genuineviral mRNAs could also be identified by these oligonucleotide sets,total nucleic acid from fibroblast cells infected with CMV AD 169 wasextracted and amplified by NASBA. All oligonucleotide sets revealedhybridization signals on Northern blot that correspond to CMV specificRNA derived amplification products. Northern blot analysis was performedas described below.

Based on ten-fold dilution series of the recombinant plasmids used forin vitro transcription of IEA RNA or pp67 RNA, the lower detection limitof the NASBA oligonucleotide pairs, when used in PCR, was about 50-100genome equivalents.

2.1.2 Detection of HCMV mRNA and DNA

Because the internal U1A mRNA control was positive in all samples, theentire series of 35 specimens was further analyzed for the presence ofthe IEA gene and its corresponding mRNA. When amplification by PCR wasperformed, eighteen samples were positive for CMV-DNA when using the IEAgene primers (Table 2). However, PCR amplification with the pp67 geneprimers failed to detect CMV-DNA in two of these eighteen samples (OT28and OT34, Table 2). This indicates that despite the fact that alreadytwo oligonucleotide pairs were used for the pp67 gene, still two sampleswere falsely negative for CMV DNA. Most likely, this is due to sequencevariation among the clinical strains in this part of the HCMV genome,since the sensitivity of the oligonucleotide sets for the IEA genetarget and the pp67 gene target are comparable.

When the samples were analyzed for IEA mRNA by NASBA amplification withthe same oligonucleotide pairs as used for DNA detection by PCR,essentially all samples that were positive by PCR were also foundpositive by NASBA. Therefore, with the exception of a single patientsample, in all specimens positive for CMV-IEA DNA the cognate mRNA couldbe detected as well.

Analysis for pp67 mRNA by NASBA revealed a strikingly different result.In contrast to IEA for which, with the exception of a single sample, allDNA-positive samples were positive for IEA-mRNA as well, pp67 mRNA couldonly be detected in a subset of the CMV-DNA positive patients (Table 2).In Table 3, the data for mRNA and DNA detection for the IEA target andthe pp67 target are summarized. When the presence or absence of pp67mRNA in these patients was correlated to their clinical status, astriking relation was observed between the presence of pp67 mRNA andpossibly CMV-related clinical symptoms (Table 4). Two patients in thisgroup showed symptoms of transplant rejection with graft dysfunction andfever. Clinical diagnoses in other patients were gastritis after hearttransplantation and retinitis in a kidney transplant recipient.Retinitis was further observed in three CMV-pp67 mRNA positive AIDSpatients, two of which also suffered from esophagitis.

                                      TABLE 1                                     __________________________________________________________________________    Primers and probes for PCR and NASBA amplificiation of CMV targets            Primer pair                                                                          Oligonucleotides                                                                       Sequence                    CMV target                                                                          SEQ ID                      __________________________________________________________________________                                                      NO.                         CMV-IEA (E4)                                                                         CMV-IEA 1.1                                                                            5'-aattctaatacgactcactatagggagaCTTGCTCACATCATGCAGCT-3'                                                    IEA-exon 4                                                                          SEQ ID NO: 1                       CMV-IEA 1.2                                                                            5'-aattctaatacgactcactatagggagaCTTGGTCACATTATAGAGTT-3'                                                          SEQ ID NO: 2                       CMV-IEA 2.1                                                                            5'-TGAGCCTTTCGAGGAGATGAA-3'       SEQ ID NO: 3                       CMV-IEA HRP-1                                                                          5'-AGGATAAGCGGGAGATGTGGAT-3'      SEQ ID NO: 4                CMV-pp67-1                                                                           CMV-pp67 1.1                                                                           5'-aattctaatacgactcactatagggagaGGGTCGATTCGAGACCGA-3'                                                      pp67  SEQ ID NO: 5                       CMV-pp67 2.1                                                                           5'-GACCTGATATCCCTCCATATA-3'       SEQ ID NO: 7                       CMV-pp67 HPR-1                                                                         5'-GGATTCGGACTTTCCGTTCGA-3'       SEQ ID NO: 8                CMV-pp67-4                                                                           CMV-pp67 1.2                                                                           5'-aattctaatacgactcactatagggagaGGGTCGATTC-AGACTGA-3'                                                      pp67  SEQ ID NO: 9                       CMV-pp67 2.1                                                                           5'-GACCTGATATCCCTCCATATA-3'       SEQ ID NO: 7                       CMV-pp67 HRP-1                                                                         5'-GGATTCGGACTTTCCGTTCGA-3'       SEQ ID NO:                  __________________________________________________________________________                                                      8                            T7 promoter sequence in downstream primers is given in small characters. 

                  TABLE 2                                                         ______________________________________                                        Analysis of CMV-IEA and CMV-pp67 DNA and mRNA in clinical                     specimens                                                                           IEA-DNA  IEA-mRNA    pp67-DNA                                                                              pp67-mRNA                                  No.   (PCR)    (NASBA)     (PCR)   (NASBA)                                    ______________________________________                                        0T01  -        -           -       -                                          0T02  ++       ++          ++      ++                                         0T03  -        -           -       -                                          0T04  ++       (+)         ++      ++                                         0T06  -        -           -       -                                          0T07  +        +           (+)     -                                          0T09  +        ++          (+)     -                                          0T10  -        -           -       -                                          0T11  -        -           -       -                                          0T12  ++       ++          ++      (+)                                        0T13  ++       ++          ++      (+)                                        0T14  ++       ++          ++      ++                                         0T16  -        -           -       -                                          0T17  ++       ++          ++      (+)                                        0T18  -        -           -       -                                          0T19  ++       ++          (+)     -                                          0T20  -        -           -       -                                          0T21  -        -           -       -                                          0T22  ++       ++          ++      ++                                         0T23  -        -           -       -                                          0T24  ++       ++          ++      ++                                         0T26  ++       ++          +       -                                          0T27  -        -           -       -                                          0T28  (+)      ++          -       -                                          0T29  ++       ++          ++      -                                          0T31  ++       ++          ++      ++                                         0T32  -        -           -       -                                          0T33  ++       +           ++      ++                                         0T34  (+)      -           -       -                                          0T35  -        -           -       -                                          0T36  -        -           -       -                                          0T37  -        -           -       -                                          0T38  -        -           -       -                                          0T39  -        -           -       -                                          0T40  (+)      ++          (+)     -                                          ______________________________________                                         No. patient sample number                                                     + positive                                                                    ++ strongly positive                                                          (+) weakly positive                                                      

                  TABLE 3                                                         ______________________________________                                                   DNA-pos/    DNA-pos/ DNA-neg/                                      CMV target RNA-pos     RNA-neg  RNA-neg                                       ______________________________________                                        IEA (n = 35)                                                                             17          1        17                                            pp67 (n = 35)                                                                             9          7        19                                            ______________________________________                                    

Two samples negative for pp67 DNA and RNA while positive for IEA DNA andRNA.

A number of CMV-positive samples do not contain detectable levels ofpp67 mRNA.

Table 4: Correlation between presence of HCMV pp67 mRNA and clinicalstatus.

    ______________________________________                                        A. pp67 DNA-pos/RNA-pos patients                                              Patient       Clinical status                                                 ______________________________________                                        Heart transplantation                                                                       graft rejection, fever, bronchial carcinoma,                    Kidney transplantation                                                                      immunocytoma                                                    Kidney transplantation                                                                      retinitis, fever                                                Kidney transplantation                                                                      graf rejection, fever                                           AIDS          retinitis, lobular hepatitis                                    AIDS          retinitis, esophagitis                                          AIDS          retinits, esophagitis                                           Heart transplantation                                                                       gastritis                                                       AIDS          epilepsy                                                        ______________________________________                                    

    ______________________________________                                        B. pp67 DNA-pos/RNA-neg patients                                              Patient       Clinical status                                                 ______________________________________                                        AIDS          Kaposi sarcoma, cryptococcus meningitis                         AIDS                                                                          Kidney transplantation                                                        Kidney transplantation                                                        Heart transplantation                                                         Heart transplantation                                                                       secundary EBV infection, immunocytoma                           Heart transplantation                                                         ______________________________________                                    

    ______________________________________                                        C. pp67 DNA-neg/RNA-neg patients                                              Patient       Clinical status                                                 ______________________________________                                        Leukemia                                                                      Liver transplantation                                                                       elevated liver enzymes                                          Heart transplantation                                                                       graft rejection                                                 Heart transplantation                                                                       graft rejection                                                 Heart transplantation                                                         AIDS          lobular pneumonia, candida infection                            --            acute myeloid leukemia, vasculitis                              Heart transplantation                                                         --            primary EBV mononucleosis                                       Kidney transplantation                                                        AIDS                                                                          Heart transplantation                                                                       graft rejection                                                 AIDS                                                                          Kidney transplantation                                                                      tuberculosis                                                    --            myelodysplastic syndrome                                        Heart transplantation                                                                       seisure                                                         Heart transplantation                                                                       sternum infection, staphylococcus aureas                        Heart transplantation                                                         Heart transplantation                                                         ______________________________________                                    

Example 2: Analytical Sensitivity of CMV-pp67 Oligonucleotides inAmplification and Detection

2.1. Materials and Methods

2.1.1. In vitro RNA

RNA with a length of 1125 nucleotides encompassing 338 nucleotides ofthe CMV mRNA encoding the pp67 matrix tegument protein was synthesizedin vitro from a cloned fragment of the corresponding gene by T7 RNApolymerase-based transcription. Prior to transcription, plasmid DNA waslinearized by Bam HI digestion, the unique restriction site of which islocated about 800 base pairs (bp) downstream of the CMV insert. Thedigested DNA was purified by phenol extraction and concentrated byethanol precipitation. Transcription from the linearized plasmid DNA wasperformed in transcription buffer [40 mM Tris-Hydrochloric acid (pH7.5); 6 mM Magnesium chloride; 2 mM Spermidine; 10 mM Sodium chloride]supplemented with 0.5 mM of each rNTP, 10 mM Dithiothreitol (DTT), 1unit per μl RNA Guard (Pharmacia), and about 500 units T7 RNA Polymerase(Pharmacia). After 4 hours incubation at 37° C., DNase I (Boehringer)was added to a final concentration of 0.1 unit per μl and the reactionmixture incubated at 37° C. for an additional 30 minutes. Subsequently,in vitro generated RNA was purified from the reaction mixture using aRNeasy RNA Purification kit (Qiagen). Finally, the concentration of thein vitro RNAs was determined by OD (260 nm) measurement and appropriateserial dilutions in water were stored at -70° C.

2.1.2. Oligonucleotides Used in Amplification and as Probes

Sequences and polarity of the oligonucleotides used in the amplificationand of the probes used for specific detection, are shown in Table 5.

All oligonucleotides were synthesized on a PCR-MATE 391 DNA synthesizer(Applied Biosystems) using phosphoramidite biochemistry.Oligonucleotides for ELGA detection (see below) were synthesized with a5'-amino link (Aminolink 2; Applied Biosystems) for subsequent couplingof horseradish peroxidase (HRP).

Oligonucleotides used in amplification were purified byelectrophoretically separating the crude oligonucleotide solutions overa 20% polyacrylamide/7M Urea slab gel and subsequent elution of thefull-length oligonucleotide from the corresponding gel band. Afterelution from the gel slices and concentration by ethanol precipitation,oligonucleotides were dissolved in Milli-Q water and concentrationsdetermined by OD(260 nm) measurement.

For ELGA detection, oligonucleotide probe CMV-pp67 HRP1 was conjugatedwith HRP (Boehringer) by coupling the enzyme to the amino link of theoligonucleotide using the cross-linking reagents SDPD (Pharmacia) andEMCS (Fluka). Unbound HRP was removed over a Qiagen Tip-100 column(Qiagen). The HRP-labeled oligonucleotide was purified by polyacrylamidegel electrophoresis and subsequent elution of the HRP-oligonucleotidefrom the gel slices by overnight incubation in water. The amount ofHRP-conjugated oligonucleotide was calculated from OD(260 nm) and OD(400nm) measurement. The solutions were stored at -70° C.

For ECL detection, oligonucleotide probe CMV-pp67-ECL (Table 5) wasconjugated with the ECL label by incubating the amino linkoligonucleotide with TAG NHS-Ester (Igen). Unbound label was removed bypassing the reaction mixture over a Qiagen Tip-100 column (Qiagen). Theamount of ECL-labelled oligonucleotide was calculated from OD (260 nm)and OD (460 nm) measurement. The solution was stored at -70° C. and usedwithout further purification.

2.1.3. NASBA Amplification

RNA amplifications were performed using the NASBA amplificationtechnology. To set up a NASBA amplification reaction, a premix wasgenerated by mixing 4 μl of 5×reaction buffer [200 mM Tris-hydrochloricacid (pH 8.5), 350 mM potassium chloride, 60 mM magnesium chloride, 25mM DTT, 5 mM of each dNTP, 10 mM of ATP, CTP and UTP, 7.5 mM of GTP and2.5 mM of ITP] with 2 μl sugar solution [15% (w/v) sucrose, 5% (w/v)mannitol, 5% (w/v) dextran T40] and 4 μl of a mix containing 1 μM ofeach oligonucleotide to be used in the amplification in 75% DMSO. Ofthis premix, 10 μl was added to 5 μl nucleic acid solution and incubatedduring 5 minutes at 65° C. Subsequently, the reaction tubes wereincubated at 41° C. during 5 minutes before 5 μl enzyme mix [32 units T7RNA polymerase; 6.4 units AMV reverse transcriptase; 0.08 unit RNase H;2.1 μg BSA; 20 mM DTT; 1.5 M sorbitol] was added. After the finaladdition, tubes were mixed by gentle tapping, centrifuged, and incubatedat 41° C. during 90 minutes. Reactions were stopped by placing them at-20° C.

2.1.4. Analysis of NASBA-amplified Reaction Products by ELGA

For the analysis of NASBA reaction products a non-radioactive enzymelinked gel assay (ELGA) based on liquid hybridization was used.Hybridization of amplification product to a specific HRP-labelledoligonucleotide probe was performed by mixing 3 μl of a NASBAamplification reaction with 1 μl 6×SSC, 1 μl concentrated loading buffer[25% (v/v) glycerol; 10 mM sodium phosphate buffer (pH 7.0); 0.05%bromophenol blue; 0.01% xylene cyanol], and 1 μl HRP-labelledoligonucleotide CMV-pp67 HRP1 (Table 5) stock solution, followed byincubation at 45° C. during 15 minutes. After hybridization, half of thereaction mixture was directly applied onto a 7% polyacrylamide gelsupplemented with 0.04% (w/v) dextran sulphate. After separation ofbound and unbound HRP-labeled oligonucleotide by electrophoresis, theprobe was visualized in the gel by direct staining with 50 ml substratesolution [125 μg 3,3',5,5'-tetramethylbenzidine per ml; 0.003% hydrogenperoxide; 100 mM sodium citrate buffer (pH 5.2)] for about 10 minutes atroom temperature. Finally, the gel was fixed by overnight incubation ina 50% (v/v) methanol solution and air dried.

2.2. Results

2.2.1. Analytical Sensitivity of CMV-pp67 Oligonucleotides When Used inNASBA

To determine the analytical sensitivity of the improved CMV-pp67 NASBApair of oligonucleotides according to the present invention (CMV-pp67-5)and to compare this sensitivity with the analytical sensitivity of theCMV-pp67-4 NASBA pair (Table 5), a dilution series of in vitro generatedRNA encompassing the target sequence of both pairs was prepared. Theindividual samples of this dilution series contained 10,000, 1,000, 100and 10 molecules of in vitro generated RNA, respectively. In severalindependent experiments NASBA amplification of this dilution series wasperformed with these two CMV-pp67 NASBA pairs of oligonucleotides (Table5). A typical example is shown in Table 6.

Direct comparison of the pairs showed that in this Example the lowestamount of in vitro RNA molecules that could be detected with CMV-pp67-4was 1,000 molecules, whereas with the improved combination CMV-pp67-5also 100 molecules of input RNA revealed a positive NASBA result.

Table 7 summarizes the results of five independent experiments. For eachpair, the lowest in vitro RNA amount still giving a positive NASBAresult in a particular experiment is shown.

The pairs that were analyzed have a comparable analytical sensitivity inNASBA, since with both combinations presence of 100 molecules of invitro generated RNA in a sample can be demonstrated upon NASBAamplification. However, in three out of five analyses the CMV-pp67-4combination appeared to have a lower detection limit of 1,000 moleculesof in vitro generated RNA, whereas CMV-pp67-5 consistently revealed apositive NASBA result for 100 molecules of input RNA in all analysesthat were performed (Table 7).

Therefore, the CMV-pp67-5 combination was regarded as more robust forNASBA amplification. It was anticipated that this advantageous aspect ofthis novel pair of oligonucleotides does not only hold true for thedetection of in vitro generated RNA, but also for detection of thegenuine CMV-pp67 protein encoding mRNA as encountered in clinicalspecimens of CMV infected individuals.

Example 3 Detection of CMV-pp67 Protein Encoding mRNA in ClinicalSamples

3 1. Materials and Methods

3.1.1. Clinical Specimens

495 samples were obtained from a cohort of 134 AIDS patients. Theseethylenediamine tetraacetic acid (EDTA) anticoagulated whole bloodsamples submitted consecutively as received by the laboratory, weremixed with nine volumes of lysis buffer [50 mM Tris-hydrochloric acid(pH 6.4); 20 mM EDTA; 1.3% (w/v) Triton X-100; 5.25 M guanidiniumthiocyanate] and stored at -70° C. until use.

3.1.2. Oligonucleotides

Sequences and polarity of the oligonucleotides used in amplification andof the probes used for specific detection, are shown in Table 5. Forfurther details is referred to paragraph 2.1.2. in the Materials andMethods section of Example 2.

3.1.3. Nucleic Acid Isolation

From the anticoagulant-treated blood specimens total nucleic acid wasisolated using guanidinium thiocyanate-mediated cell lysis andadsorption of nucleic acid to silica particles (Boom et al., J. of Clin.Microbiol. 28, 495-503, 1990).

Whole blood samples in Lysis buffer were thawed and from each sample 1ml (equivalent to 100 μl whole blood) was transferred into an Eppendorftube. Subsequently, 50 μl of hydrochloric acid-activated silicum dioxideparticles [size-selected suspension of 1 mg/ml in 0.1 M hydrochloricacid (Sigma); see ref. Boom et al., 1990] were added and the suspensionwas incubated during 10 minutes at room temperature with regularvortexing. Nucleic acid bound to the silica was spun down bycentrifugation. Pelleted silica particles were washed twice with 1 mlGuSCN wash buffer [50 mM Tris-hydrochloric acid (pH 6.4); 5.25 Mguanidinium thiocyanate], followed by two washing steps with 1 ml 70%ethanol and a single washing step with 1 ml acetone. After each washingstep, the suspension was briefly centrifuged and the silica pellet wasresuspended in the next washing solution by thorough mixing. Afterremoval of the acetone, the silica particles were dried by incubation at56° C. in a heating block during 10 minutes. Nucleic acid was elutedfrom the silica particles by incubation in 50 μl Tris-buffered elutionmedium (pH 7.5) at 56° C. during 10 minutes Finally, the silicaparticles were spun down again and the supernatant was carefullypipetted into fresh reaction tubes avoiding any carry-over of silica.Extracted nucleic acid samples were stored at -70° C. until use.

3.1.4. NASBA Amplification

For amplification, a so-called accusphere containing all the ingredientsnecessary for a NASBA reaction (see "NASBA amplification" paragraph inthe Materials and Methods section of Example 2) in a lyophilized form,was reconstituted in a Tris-HCl (pH 8.5) buffered solution of 30% (v/v)DMSO. Subsequently, for each sample to be analysed, 5 μl nucleic acidsolution and 10 μl of the oligonucleotide solution were added to a testtube. The resulting mixtures were heated at 65° C. for 5 minutes, afterwhich the tubes were placed at 41° C. After 5 minutes incubation at 41°C., 5 μl of enzyme solution containing 32 units T7 RNA polymerase, 6.4units of AMV reverse transcriptase and 0.08 unit RNase H were added andthe contents of the tube were mixed by gentle tapping. The reactionswere incubated at 41° C. for 90 minutes in a water bath. Reactions werestopped by placing them at -20° C.

3.1.5. Analysis of NASBA Amplified Reaction Products by ECL Detection

For electrochemiluminescence (ECL) based analysis, detection reagentswere prepared by vortexing a capture probe solution, containingbiotinylated capture probe CMV-pp67 CAP (Table 5) immobilized onstreptavidin-coated paramagnetic beads (Dynal), until an opaque solutionwas formed and subsequently mixing 10 μl of this suspension (containing3.4×105 beads loaded with capture probe) and 10 μl of a CMV-pp67-ECL(Table 5) probe solution (containing 3.4×1011 molecules of ECL labeledprobe) into fresh reaction tubes. To these mixtures, 5 μl of a two-folddiluted NASBA reaction was added and incubated during 30 minutes at 41°C. During hybridization, the tubes were mixed every 10 minutes.Subsequently, 300 μl NASBA QR System Assay buffer (organon Teknika) wereadded to each hybridization tube and the tubes were positioned in aNASBA QR System for automated reading of ECL signals.

3.2. Results

3.2.1. Detection of CMV-pp67 mRNA in Clinical Samples with Two DifferentPairs of Oligonucleotides

Whole blood samples from patients clinically at risk of infection withCMV were analysed for the presence of CMV mRNA encoding the matrixtegument protein pp67 (CMV-pp67 mRNA) with the two distinct pairs ofoligonucleotides (Table 5) suited for NASBA amplification.

In total, 495 samples from AIDS patients, previously analysed by NASBAamplification with combination CMV-pp67-4 (Table 5), now were reanalyzedunder similar NASBA conditions with the improved pair CMV-pp67-5 whichis the object of the present invention. In Table 8, the results obtainedwith either of the pairs are shown and compared to each other.

Although from earlier experiments no (statistically) significantdifference in the analytical sensitivity of these sets ofoligonucleotides could be determined (due to the fact that only a fewsamples were tested; see Example 2), the improved CMV-pp67-5 setappeared to be much better for the detection of CMV-pp67 mRNA by NASBAin nucleic acid solutions extracted from whole blood samples. Due to theamount of samples (n=495), this difference is also statisticalsignificant.

In 65 samples, the improved pair CMV-pp67-5 revealed a positive NASBAresult, whereas NASBA reactions performed with the CMV-pp67-4 pair werenegative. In only 9 cases (of which 5 samples are difficult tointerpret) it was the other way around.

In conclusion, apart from being more robust than the CMV-pp67-4 set (seeExample 2), the novel and improved pair of oligonucleotides according tothe present invention (CMV-pp67-5) also appears to be superior in thedetection of CMV-pp67 mRNA in clinical samples.

                                      TABLE 5                                     __________________________________________________________________________    Oligonucleotides used in NASBA amplification and                              detection of CMV-pp67 mRNA                                                    Pair   Oligonucleotides                                                                      SEQ ID NO:                                                                            Sequence                                               __________________________________________________________________________    CMV-pp67-4                                                                           CMV-pp67 P1.2                                                                         SEQ ID NO: 9                                                                          5'-aattctaatacgactcactatagggagaGGGTCGATTCAGACTGA-3'           CMV-pp67 P2.1                                                                         SEQ ID NO: 7                                                                          5'-GACCTGATATCCCTCCATATA-3'                                   CMV-pp67 HRP1                                                                         SEQ ID NO: 8                                                                          5'-GGATTCGGACTTTCCGTTCGA-3'                            CMV-pp67-5                                                                           CMV-pp67 P1.2                                                                         SEQ ID NO: 9                                                                          5'-aattctaatacgactcactatagggagaGGGTCGATTCAGACTGA-3'           CMV-pp67 P2.4                                                                         SEQ ID NO: 11                                                                         5'-CTGGAGATATATGTTGACCA-3'                                    CMV-pp67-CAP                                                                          SEQ ID NO: 8                                                                          5'-BIO-GGATTCGGACTTTCCGTTCGA-3'                               CMV-pp67-ECL                                                                          SEQ ID NO: 12                                                                         5'-ECL-CCAAAAAGCTAGCCGTCACG-3'                         __________________________________________________________________________     The T7 promoter is given in small letters.                               

                  TABLE 6                                                         ______________________________________                                        Analytical sensitivity of two CMV-pp67 pairs of                               oligonucleotides when used in NASBA.                                                      ELGA result                                                       Input RNA     CMV-pp67-4 CMV-pp67-5                                           ______________________________________                                        10.sup.4      +          +                                                    10.sup.3      +          +                                                    10.sup.2      -          +                                                    10.sup.1      -          -                                                    ______________________________________                                    

                  TABLE 7                                                         ______________________________________                                        Comparison of two CMV-pp67 pairs of oligonucleotides                          used in NASBA.                                                                           Lower detection limit*                                             Experiment   CMV-pp67-4 CMV-pp67-5                                            ______________________________________                                        1            10.sup.3   10.sup.2                                              2            10.sup.2   10.sup.2                                              3            10.sup.2   10.sup.2                                              4            10.sup.2   10.sup.2                                              5            10.sup.3   10.sup.2                                              ______________________________________                                         *Lower detection limit: lowest amount of input RNA that still reveals a       positive NASBA result                                                    

                  TABLE 8                                                         ______________________________________                                        Analysis of CMV-pp67 mRNA oligonucleotides in                                 amplifications performed on clinical specimens (n = 495)                                      novel CMV-pp67-5 oligo's                                                      positive                                                                              negative                                              ______________________________________                                        (known)     positive  53(10)*   4(5)*                                         CMV-pp67-4  negative  65        358                                           oligo's                                                                       ______________________________________                                         *between brackets the number of weakly positive samples obtained with the     CMVpp67-4 pair is given                                                  

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 12                                            - (2) INFORMATION FOR SEQ ID NO: 1:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 48 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                               -     (ii) MOLECULE TYPE: cDNA to mRNA                                        #1:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   #                48ACTA TAGGGAGACT TGCTCACATC ATGCAGCT                        - (2) INFORMATION FOR SEQ ID NO: 2:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 48 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                               -     (ii) MOLECULE TYPE: cDNA to mRNA                                        #2:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   #                48ACTA TAGGGAGACT TGGTCACATT ATAGAGTT                        - (2) INFORMATION FOR SEQ ID NO: 3:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 21 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                               -     (ii) MOLECULE TYPE: cDNA to mRNA                                        #3:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   #21                ATGA A                                                     - (2) INFORMATION FOR SEQ ID NO: 4:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 22 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                               -     (ii) MOLECULE TYPE: cDNA to mRNA                                        #4:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   #                 22TGG AT                                                    - (2) INFORMATION FOR SEQ ID NO: 5:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 46 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                               -     (ii) MOLECULE TYPE: cDNA to mRNA                                        #5:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   #                 46CTA TAGGGAGAGG GTCGATTCGA GACCGA                          - (2) INFORMATION FOR SEQ ID NO: 6:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 18 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                               -     (ii) MOLECULE TYPE: cDNA to mRNA                                        #6:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   #  18              GA                                                         - (2) INFORMATION FOR SEQ ID NO: 7:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 21 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                               -     (ii) MOLECULE TYPE: cDNA to mRNA                                        #7:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   #21                ATAT A                                                     - (2) INFORMATION FOR SEQ ID NO: 8:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 21 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                               -     (ii) MOLECULE TYPE: cDNA to mRNA                                        #8:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   #21                TTCG A                                                     - (2) INFORMATION FOR SEQ ID NO: 9:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 45 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                               -     (ii) MOLECULE TYPE: cDNA to mRNA                                        #9:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   #45                ACTA TAGGGAGAGG GTCGATTCAG ACTGA                           - (2) INFORMATION FOR SEQ ID NO: 10:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 17 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                               -     (ii) MOLECULE TYPE: cDNA to mRNA                                        #10:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   #   17             A                                                          - (2) INFORMATION FOR SEQ ID NO: 11:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                               -     (ii) MOLECULE TYPE: cDNA to mRNA                                        -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Cytomegalovi - #rus                                   #11:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   # 20               ACCA                                                       - (2) INFORMATION FOR SEQ ID NO: 12:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                               -     (ii) MOLECULE TYPE: cDNA to mRNA                                        -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Cytomegalovi - #rus                                   #12:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   #  20              CACG                                                       __________________________________________________________________________

We claim:
 1. An oligonucleotide which is a subsequence of a nucleic acidsequence encoding human cytomegalovirus pp 67, said oligonucleotidebeing 10-35 nucleotides in length and comprising at least 10 consecutivenucleotides of SEQ ID NO:10, said oligonucleotide covalently bonded toan RNA polymerase promoter sequence.
 2. The oligonucleotide according toclaim 1, wherein the promoter sequence is the T7 RNA polymerase promotersequence.
 3. A pair of oligonucleotides for the amplification of atarget sequence located within a human cytomegalovirus pp67 sequence, afirst oligonucleotide of said pair being 10-35 nucleotides in length andcomprising at least 10 consecutive nucleotides of the nucleic acidsequence (SEQ ID NO:10) and a second oligonucleotide of said pair beingof 10-35 nucleotides in length and comprising at least 10 consecutivenucleotides of the nucleic acid sequence (SEQ ID NO:11).
 4. A method forthe diagnosis of symptomatic cytomegalovirus disease, wherein thepresence of pp67 mRNA encoding a late structural protein of the humancytomegalovirus in a blood sample of an individual suspected of carryingsaid disease is detected, comprising:amplifying a target sequence withinsaid mRNA using a pair of oligonucleotides according to claim 3 andamplification reagents, reacting the sample, optionally containingamplified nucleic acid, with an oligonucleotide probe, and detectinghybrids formed between the target sequence and the probe.
 5. The methodaccording to claim 4, wherein the first oligonucleotide of said pair ofoligonucleotides is covalently bonded to an RNA polymerase promotersequence.
 6. The method according to claim 4, wherein theoligonucleotide probe, or its complement, is 10-35 nucleotides in lengthand comprises at least 10 consecutive nucleotides of a sequence selectedfrom the group consisting of: 5'-GGATTCGGACTTTCCGTTCGA-3' (SEQ ID NO: 8)and 5'-CCAAAAAGCTAGCCGTCACG-3' (SEO ID NO:12), and which is providedwith a detectable label.
 7. The method according to claim 4, wherein themRNA is amplified using a transcription based amplification technique.8. The method according to claim 7, wherein said amplification techniqueis nucleic acid sequence based amplification (NASBA).
 9. The pair ofoligonucleotides according to claim 3, wherein the first oligonucleotideis SEQ ID NO:10 and the second oligonucleotide is SEQ ID NO:11.
 10. Thepair of oligonucleotides according to claim 9, wherein the firstoligonucleotide is covalently bonded to an RNA polymerase promotersequence.
 11. The pair of oligonucleotides according to claim 10,wherein the promoter sequence is the T7 RNA polymerase promotersequence.
 12. The pair of oligonucleotides according to claim 3, whereinthe first oligonucleotide is covalently bonded to an RNA polymerasepromoter sequence.
 13. A test kit for the diagnosis of humancytomegalovirus disease, comprising a pair of oligonucleotides for theamplification of a target sequence located within a humancytomegalovirus pp67 sequence, a first oligonucleotide of said pairbeing 10-35 nucleotides in length and comprising at least 10 consecutivenucleotides of the nucleic acid sequence 5'-GGGTCGATTCAGACTGA-3' (SEQ IDNO:10) and a second oligonucleotide of said pair being 10-35 nucleotidesin length and comprising at least 10 consecutive nucleotides of thenucleic acid sequence 5'-CTGGAGATATATGTTGACCA-3' (SEQ ID NO:11), saidtest kit further comprising an oligonucleotide probe, which is providedwith a detectable label.
 14. The test kit of claim 13, wherein saidfirst oligonucleotide is covalently bonded to an RNA polymerase promotersequence.
 15. The test kit of claim 14, wherein the promoter sequence isthe T7 RNA polymerase promoter sequence.
 16. The test kit of claim 13,wherein said oligonucleotide probe comprises a nucleic acid sequenceselected from the group consisting of: 5'-GGATTCGGACTTTCCGTTCGA-3' (SEQID NO:8) and 5'CCAAAAAGCTAGCGTCACG-3' (SEQ ID NO:12).
 17. The pair ofoligonucleotides according to claim 12, wherein the promoter sequence isthe T7 RNA polymerase promoter sequence.
 18. The method according toclaim 5, wherein the promoter sequence is the T7 RNA polymerase promotersequence.